Study Of Regulation Mechanism For EGF Is Involved In The Follicular Development And Granulosa Cell Proliferation In Muscovy Duck

Muscovy duck and common domestic duck belongs to a different genus and have the characteristics of strong broodiness and low egg-laying performance.In order to provide a theoretical basis for the reduction of broodiness in Muscovy duck.The study mainly consists of four parts,exploring the relevant mechanism of EGF involved in follicular development and granulosa cell proliferation,and further exploring the mechanism of EGF mediated FSH involved in the pregrade follicular granulosa cells proliferation in Muscovy duck.The main results are as follows:1.The morphological changes in the growth and development of Muscovy duck follicles were studied,and it was found that the compared with the laying period,the ovaries presented the phenomena of follicular atrophy,cytoplasmic concentration and unclear boundary;in all levels of follicles development,the granulosa cell layer thickness reached its peak in SYF period,and the membrane layer thickness generally increased with the development of follicles that reached its peak in F3 period;to all levels of the follicle hormone levels were measured and found FSH appeared two peaks(LYF and F4 periods,respectively is 83.33±1.31 m IU/g and70.56±0.93 m IU/g),LH also presents a similar trend(LYF and F4 periods are peak),P4 and E2 concentration along with the development of follicle showed a trend of gradually reduce the overall and enter grade follicle period to maintain in a relatively stable and lower level;The content of EGF in SYF and LYF period were significantly higher than other periods(P<0.05),59.06 ng/g and 57.58 ng/g respectively;the expression levels of EGF and EGFR m RNA in all levels of follicles reached a peak in SYF and LYF periods were significantly higher than other periods(P<0.05).2.In vitro culture model of pregrade follicular granulosa cells in Muscovy ducks was constructed,by adopting the combination of mechanical separation and enzyme separation method to separate granulosa cells(with 0.1% type Ⅱ collagenase digestion in incubator 8-10 min,1800 rpm centrifugal 5 min).M199 complete culture medium containing 0.5% FBS was cultured for 24 h and then changed to serumfree culture medium for subsequent experiments a better results can be obtained.3.To investigate the regulation mechanism of EGF on the proliferation of pregrade follicular granulosa cells in Muscovy duck.Treatment of granulosa cells with different concentrations of EGF and FSH showed that the proliferation intensity of EGF on granulosa cells was positively correlated with the dose,and combined treatment of granulosa cells with EGF(10 ng/m L)and FSH(50 m IU/m L)showed the most significant proliferation at 24 h(P<0.05),with the best effect;In vitro suspension culture of pregrade follicles,the thickness of granulosa cell layer and membrane layer increased after EGF(10 ng/m L)and FSH(50m IU/m L)were treated alone or in combination for 24 h,and the increase was the most significant when treated in combination(P<0.05);after the addition of 10 ng/m L EGF treatment cells for 24 h,the m RNA expression level of cell cycle genes(CCND1,CCNE1,CDK2,CDK6)was significantly higher than that of the control group(P<0.05),the expression level of anti-apoptotic gene Bcl-2 m RNA was significantly increased(P<0.05),and the expression level of pro-apoptotic gene Bax m RNA was significantly decreased(P<0.05);after the treatment of granulosa cell with Gefitinib,the cell proliferation activity significantly decreased in the nontoxic range and showed a concentration-dependent characteristic,and with the increase of treatment time,the cell activity significantly decreased,showing a time-dependent effect;overexpression of EGFR significantly increased the cell number and the expression levels of CCND1,CCNE1,CDK2 and CDK6 genes were significant rise(P<0.05),in which the expression level of CDK2 increased the most,followed by the significant increase of Bcl-2 gene(P<0.05),and the expression level of Bax gene was significantly decreased(P<0.05),and also shows that EGF promotes cell proliferation inhibition of cell apoptosis.4.The mi RNA sequencing was performed on the ovaries of Muscovy duck in the broody and laying periods using Solexa technology.A total of22 differentially expressed mi RNAs that are newly predicted mi RNA,among which 7 were up-regulated and 15 were down-regulated(the laying group as the control group).The target genes prediction,GO functional annotation and KEGG pathway analysis were performed on differentially expressed mi RNAs.It found that the EGFR and EGF were one of the target genes of unconservative＿4＿17575 and unconservative＿pedo01007066.1＿48822,respectively;the MAPK signaling pathway,Rap1 signaling pathway and Gap junction pathway were significantly enriched pathways for differentially expressed mi RNAs and were associated with follicular development and granulosa cell proliferation;the unconservative＿pedo01000238.1＿42849,unconservative＿4＿17575,unconservative＿2＿9994,unconservative＿5＿19313 and unconservative＿pedo01007066.1＿48822through respectively targeted regulation including CASP3,STK3,CDK1,RAPGEF4,MAP3K4,DOCK4 and GRB2 play an important role in these three pathways.The expression levels of 6 differentially expressed mi RNA was determine after treatment 10 ng/m L EGF for 24 h by q RT-PCR.Compared with the control group,the expression levels of unconservative＿pedo01000238.1＿42849 and unconservative＿4＿17575were significantly decreased(P<0.01).